Document Type : Original Article


1 Department of Clinical Sciences, Faculty of Veterinary Medicine, Amol University of Special Modern Technologies, Amol, Iran

2 Iran Simmental Cattle Breeding Center, Amard-dam Company, Amol, Iran


Artificial insemination is a well-established and widely used method for genetic improvement in cattle breeding industry. Recently, researchers have shown an increased interest in the cryoprotective effects of minerals and antioxidants on semen. Previous studies on calcium (Ca) and magnesium (Mg), two main macro-minerals, have mainly investigated their roles in mammalian spermatogenesis and fertility. In addition, the experimental data examining the semen content regarding these minerals and antioxidants from different animal species are rather controversial and there is no general agreement about their associations with semen quality. Therefore, this study was conducted to assess the seminal plasma concentrations of Ca, Mg and total antioxidant capacity (TAC) in first and second ejaculations of dual-purpose Fleckvieh bulls and to link them to the sperm characteristics of fresh and frozen-thawed semen. Sperm progressive motility after thawing was used to classify the data into three groups: < 40.00%, 40.00 to 50.00% and > 50.00%. The measurements of two minerals and TAC were carried out using spectrophotometry and enzyme-linked immunosorbent assays, respectively. The results showed that there were significant differences in several parameters of semen quality between first and second ejaculations. No significant differences were also found on Ca and Mg concentrations and Ca/Mg ratio. The TAC level was significantly higher in the first ejaculation than the second one. The findings of this study suggest that TAC is a potential marker for bull semen quality assessment in the frozen semen production industry.


Main Subjects


    1. Frankel MS. Cryobanking of human sperm. J Med Ethics 1975; 1(1): 36-38.
    2. Li Y, Kalo D, Zeron Y, et al. Progressive motility-a potential predictive parameter for semen fertilization capacity in bovines. Zygote 2016; 24(1): 70-82.
    3. Ibanescu I, Leiding C, Ciornei ŞG, et al. Differences in CASA output according to the chamber type when analyzing frozen-thawed bull sperm. Anim Reprod Sci 2016; 166: 72-79.
    4. Caballero I, Parrilla I, Alminana C, et al. Seminal plasma proteins as modulators of the sperm function and their application in sperm biotechnologies. Reprod Domest Anim 2012; 47(S3): 12-21.
    5. Talluri TR, Mal G, Ravi SK. Biochemical components of seminal plasma and their correlation to the fresh seminal characteristics in Marwari stallions and Poitou jacks. Vet World 2017; 10(2): 214-220.
    6. Hamamah S, Gatti JL. Role of the ionic environment and internal pH on sperm activity. Hum Reprod 1998; 13(S4): 20-30.
    7. Tvrdá E, Lukac N, Schneidgenova M, et al. Impact of seminal chemical elements on the oxidative balance in bovine seminal plasma and spermatozoa. J Vet Med 2013; 125096:
    8. Pipan MZ, Mrkun J, Strajn BJ, et al. The influence of macro-and microelements in seminal plasma on diluted boar sperm quality. Acta Vet Scand 2017; 59(1): 11. doi.10.1186/s13028-017-0279-y.
    9. Meseguer M, Garrido N, Martínez-Conejero JA, et al. Role of cholesterol, calcium, and mitochondrial activity in the susceptibility for cryodamage after a cycle of freezing and thawing. Fertil Steril 2004; 81(3):
    10. Fraser LR. Minimum and maximum extracellular Ca2+ requirements during mouse sperm capacitation and fertilization in vitro. J Reprod Fertil 1987; 81(1): 77-89.
    11. Henricks DM. Biochemistry and physiology of the gonadal hormones. In: Cupps PT (Eds). Reproduction in domestic animals. 4th ed. California USA: Elsevier 1991; 76-117.
    12. Juyena NS, Stelletta C. Seminal plasma: An essential attribute to spermatozoa. J Androl 2012; 33(4):
    13. Yasothai R. Importance of minerals on reproduction in dairy cattle. Int J Sci Environ Technol 2014; 3(6): 2051-2057.
    14. Abdel-Rahman H, El-Belely MS, Al-Qarawi AA, et al. The relationship between semen quality and mineral composition of semen in various ram breeds. Small Rumin Res 2000; 38(1): 45-49.
    15. Anel-Lopez L, Ortega-Ferrusola C, Martínez-Rodríguez C, et al. Analysis of seminal plasma from brown bear (Ursus arctos) during the breeding season: Its relation-ship with testosterone levels. PloS One 2017; 12(8): e0181776.
    16. Yazdani M. Concerns in the application of fluorescent probes DCDHF-DA, DHR 123 and DHE to measure reactive oxygen species in vitro. Toxicol In Vitro 2015; 30(1 Pt B): 578-582.
    17. Tremellen K. Oxidative stress and male infertility-A clinical perspective. Hum Reprod Update 2008; 14(3): 243-258.
    18. Pujol A, Obradors A, Esteo E, et al. Oxidative stress level in fresh ejaculate is not related to semen parameters or to pregnancy rates in cycles with donor oocytes. J Assist Reprod Genet 2016; 33(4): 529-534.
    19. Smith R, Vantman D, Ponce J, et al. Andrology: Total antioxidant capacity of human seminal plasma. Hum Reprod 1996; 11(8): 1655-1660.
    20. Gurler H, Calisici O, Bollwein H. Inter-and intra-individual variability of total antioxidant capacity of bovine seminal plasma and relationships with sperm quality before and after cryopreservation. Anim Reprod Sci 2015; 155: 99-105.
    21. Eghbali M, Alavi-Shoushtari SM, Asri-Rezaei S, et al. Calcium, magnesium and total antioxidant capacity (TAC) in seminal plasma of water buffalo (Bubalus Bubalis) bulls and their relationships with semen characteristics. Vet Res Forum 2010; 1(1): 12-20.
    22. Arver S. Zinc and zinc ligands in human seminal plasma III. The principal low molecular weight zinc ligand in prostatic secretion and plasma seminal. Acta Physiol Scand 1982; 116(1): 67-73.
    23. Mortimer D. Practical laboratory andrology. New York, USA: Oxford University Press 1994; 89–109.
    24. Kanno C, Sakamoto KQ, Yanagawa Y, et al. Comparison of sperm subpopulation structures in first and second ejaculated semen from Japanese black bulls by a cluster analysis of sperm motility evaluated by a CASA system. J Vet Med Sci 2017; 79(8): 1359-1365.
    25. Frangez R, Gider T, Kosec M. Frequency of boar ejaculate collection and its influence on semen quality, pregnancy rate and litter size. Acta Vet Brno 2005; 74(2): 265-273.
    26. Valsa J, Skandhan KP, Sumangala B, et al. Time bound changes (in 24 h) in human sperm motility and level of calcium and magnesium in seminal plasma. Alexandria J Med 2016; 52(3): 235-241.
    27. Hebles M, Dorado M, Gallardo M, et al. Seminal quality in the first fraction of ejaculate. Syst Biol Reprod Med 2015; 61(2): 113-116.
    28. Carvalho LE, Silva Filho JM, Palhares MS, et al. Physical and morphological characteristics of the first three jets of Pega jackasses sperm-rich fraction. Arq Bras Med Vet Zootec 2016; 68(4): 845-852.
    29. Axnér E, Strom B, Linde-Forsberg C. Sperm morphology is better in the second ejaculate than in the first in domestic cats electroejaculated twice during the same period of anesthesia. Theriogenology 1997; 47(4): 929-934.
    30. Farrell P, Presicce GA, Brockett CC, et al. Quantification of bull sperm characteristics measured by computer-assisted sperm analysis (CASA) and the relationship to fertility. Theriogenology 1998; 49(4): 871-879.
    31. Gopinathan A, Sivaselvam SN, Karthickeyan SK, et al. Influence of non-genetic factors on semen quality parameters in crossbred Jersey (Bos taurus x Bos indicus) bulls. Int J Curr Microbiol App Sci 2018; 7(4): 2994-3004.
    32. Kirton KT, Hafs HD, Hunter AG. Levels of some normal constituents of bull semen during repetitive ejaculation. J Reprod Fertil 1964; 8(2):157-164.
    33. Pickett BW, Komarek RJ. Lipid and dry weight of bovine seminal plasma and spermatozoa from first and second ejaculates. J Dairy Sci 1967; 50(5): 742-746.
    34. Everett RW, Bean B. Environmental influences on semen output. J Dairy Sci 1982; 65(7): 1303-1310.
    35. Davis HP, Williams NK. Evaluating bovine semen I. Influence of the number of ejaculates upon various physical and chemical characteristics and the relationship between those factors. J Anim Sci 1939; 1: 232-242.
    36. Nel-Themaat L, Harding GD, Chandler JE, et al. Quality and freezing qualities of first and second ejaculates collected from endangered Gulf Coast Native rams. Anim Reprod Sci 2006; 95(3-4): 251-261.
    37. Kanwal, MR., Rehman NU, Ahmad N, et al. Bulk cations and trace elements in the Nili-Ravi buffalo and crossbred cow bull semen. Int J Agric Biol 2000; 2(4): 302-305.
    38. Mrackova M, Zavadilova M, Sedlinska M. Assessment of the effect of selected components of equine seminal plasma on semen freezability. Mac Vet Rev 2015; 38(1): 91-96.
    39. Usuga A, Rojano B, Restrepo G. Effect of seminal plasma components on the quality of fresh and cryopreserved stallion semen. J Equine Vet Sci 2017; 58: 103-111.
    40. Wong WY, Flik G, Groenen PM, et al. The impact of calcium, magnesium, zinc, and copper in blood and seminal plasma on semen parameters in men. Reprod Toxicol 2001; 15(2): 131-136.
    41. Quinn PJ, White IG, Wirrick BR. Studies of the distribution of the major cations in semen and male accessory secretions. J Reprod Fertil 1965; 10(3): 379-388.
    42. Ghaniei A, Eslami M, Babaei Marzango SS. Determination of calcium, magnesium, phosphorus, iron, and copper contents in rooster seminal plasma and their effects on semen quality. Comp Clin Path 2018; 27(2): 427-431.
    43. Zaja IZ, Samardzija M, Vince S, et al. Influence of boar breeds or hybrid genetic composition on semen quality and seminal plasma biochemical variables. Anim Reprod Sci 2016; 164: 169-176.
    44. Gur S, Demirci E. Effect of calcium, magnesium, sodium and potassium levels in seminal plasma of holstein bulls on spermatological characters. Turk J Vet Anim Sci 2000; 24(3): 275-281.
    45. Garcia MA, Graham EF. Development of a buffer system for dialysis of bovine spermatozoa before freezing. III. Effect of different inorganic and organic salts on fresh and frozen-thawed semen. Theriogenology 1989; 31(5): 1039-1048.
    46. Pesch S, Bergmann M, Bostedt H. Determination of some enzymes and macro-and microelements in stallion seminal plasma and their correlations to semen quality. Theriogenology 2006; 66(2): 307-313.
    47. Bozkurt Y, Ogretmen F, Kokcu O, et al. Relationships between seminal plasma composition and sperm quality parameters of the Salmo trutta macrostigma (Dumeril, 1858) semen: with emphasis on sperm motility. Czech J Anim Sci 2011; 56(8): 355-364.
    48. Kaya A, Aksoy M, Tekeli T. Influence of ejaculation frequency on sperm characteristics, ionic composition and enzymatic activity of seminal plasma in rams. Small Rumin Res 2002; 44(2): 153-158.
    49. Barrier-Battut I, Delajarraud H, Legrand E, et al. Calcium, magnesium, copper, and zinc in seminal plasma of fertile stallions, and their relationship with semen freezability. Theriogenology 2002; 58(2-4): 229-232.
    50. Liang H, Miao M, Chen J, et al. The association between calcium, magnesium, and ratio of calcium/magnesium in seminal plasma and sperm quality. Biol Trace Elem Res 2016; 174(1): 1-7.